Adenoviruses (Ad) are major causative agents of respiratory, ocular and enteric diseases. Replication-defective and conditionally replicating Ad vectors are also being employed in approximately 25% of human gene transfer as well as for the development of anti-microbial vaccines. Further progress in these areas is currently hampered by the lack of accurate structural information on intact Ad particles. While there are crystal structures for several of the major Ad capsid proteins (hexon, penton base and fiber), we lack detailed knowledge of their association upon assembly into intact Ad particles. Moreover, the precise location and structure of nine other capsid proteins as well as the organization of the Ad genome in the virion core are unknown. In particular, we lack structural information on a key capsid protein that mediates endosome disruption during virus entry (Wiethoff et al., 2005). Therefore, the major goal of this proposal is to determine the structure of human adenovirus at near atomic resolution using x-ray crystallography. The topology and fold of the major and minor capsid proteins and their arrangement in wild type as well as in a hyperstable/non-infectious mutant Ad will be investigated. This information should increase the knowledge of the underlying interactions that influence virus assembly/disassembly and cell entry. Furthermore, detailed knowledge on the structure of human adenovirus may facilitate the re-engineering of adenoviral vectors for gene transfer or vaccine development. [unreadable] [unreadable] Recently, we were successful in obtaining good diffraction quality crystals of human adenovirus. Crystallographically, this project has been and continues to be challenging due to the size and complexity of the Ad particle (about 900A in diameter and approximately 150 MDa). To our knowledge, this is the largest macromolecular complex successfully attempted to be solved by x-ray diffraction. This is reflected by large unit cell dimensions (1485, 892, 878, 90, 125.5, 90; space group: C2). The current form of the Ad crystals diffract to 4-5A resolution. The knowledge and expertise acquired by analyzing the structure of Ad by x-ray diffraction may facilitate the structural analysis of even larger and more complex icosahedral viruses that are beginning to emerge (Wilson, W. et al. Science 309: 1090-1092, 2005). [unreadable] [unreadable] [unreadable]